Construction And Identification Of P815Cell Line Stably Expressing Chicken B-LB

Major histocompatibility complex(MHC), a genic system located in chromosomewhich makes of a group of close linkage and highly polymorphism gene locus, which wasgenerally found in almost all vertebrates. MHC molecules are also considered as theprimary determinants in the transplant rejection, immune respond and immune regulation.According to their structures and functions, MHC molecules could be divided into classⅠ,Ⅱ and Ⅲ.Class Ⅱ molecules are glycoproteins that are formed by twononcovalence-associated chains. Class Ⅱ molecules are consisted of peptide-bindingregion, immunoglobulin-like region, transmenbrane region and cytoplasmic region. Wecalled the major histocompatibility complex of chicken B-complex, which locates in16thmicro-chromosome. According to the protein nature, the chicken MHC molecules couldalso be divided into B-F, B-L, B-G. and B-L. These molecules play an important roleduring the process of exogenous antigen presenting, which have certain homology withMHC molecules in mammalian. The gene encoding B-L molecules contain B-LA whichencods the α chain and B-LB which encodes the β chain. This research for the task is tostudy B-LB.MHC class Ⅱ molecules mainly distribute on the membrane of the maturing B cells,antigen presenting cells (macrophage cells and dendritic cells) and some activated T cells.These molecules have an effect on presenting antigen and activating T cells by formingclassⅡmolecule-antigen peptides complex, which could stimulate T cells and B cells toparticipate in immune response. In order to research the function and mechanism of classMHCⅡ-of chicken in immune response, we cloned, expressed and identified the gene ofchain of class Ⅱ by using molecular biological methods and cytobiological methods,and also successfully constructed the P815cell line stably expressing chicken-MHCⅡ-.First, according to the mRNA sequence of chicken MHCⅡ β and multi-cloningrestriction enzyme sites of vector pEFGP-N1, a pair of specific primers for the gene ofchain of class Ⅱ of chicken was designed and synthesized. At the same time, accordingto the gene fragment of the chicken MHCⅡ β gene that was from332bp to792bp, a pairof primers which was used to detect the cell line stablity was designed and synthesized aswell. Using total RNA from chicken spleen lymphocytes, a gene fragment about792bp was amplified by RT-PCR. The result of special endonuclease digesting RT-PCR productof chain showed that the gene had the corresponding restriction enzyme sites as theknown gene. Then the fragment was inserted into vector pEFGP-N1. We transientlytransfected pEGFP-N1/B-LB to COS7cell line and stably transfected P815cell line. Weobserved the result of transient transfection using fluorescence microscope and found thefluorescent light of GFP-MHCⅡ-distributed on cell endomembrane system.Under thepressure of G418, we obtained the cell line P815-MHCⅡ-which could stably expressGFP-MHCⅡ-by using special primers and RT-PCR detection.In this research the recombinant plasmid pEGFP-N1/B-LB was successfullyconstructed and P815cell line stably expressing chicken-MHCⅡ-was acquired. Thetumor cell model could provide a tool for testing and evaluating the efficacy of anti-tumorimmunological drugs targeting MHCⅡ-, and laid foundation for further research onMHCⅡ-.